Anti-blastocystosis activity of antioxidant coated ZIF-8 combined with mesoporous silicas MCM-41 and KIT-6

The biocompatible hybrid Zeolitic imidazolate framework-8 (ZIF-8)/structured silica nanocomposite can be loaded with antioxidants such as curcumin and resveratrol to offer multiple advantages of drug functionalization and structural stability. blastocystosis, an enteric parasite, has various outcomes and its treatment includes drugs which have side effects and do not result in a full cure. We aimed to design novel biocompatible nanocomposites containing natural antioxidant, resveratrol or curcumin and ZIF-8/mesoporous silica. We also assessed their anti-blastocystosis activities as bioactive novel nanocomposites. The nano-silica (MCM-41 and KIT-6) was synthesized using a hydrothermal technique and made composite with ZIF-8 using an ultrasonic technique. The antioxidants, curcumin and resveratrol, were loaded over ZIF-8/MCM-41 and ZIF-8/KIT-6 using a rotary evaporator technique to form novel nanocomposites with bioactive properties. The formulated nanocomposites were characterized. To test their biological activity, suspension of cultured blastocystosis cysts (subtype 3) were exposed to increasing concentrations of nanocomposites and the minimal lethal concentration of each nanocomposite was calculated. The bioactive nanocomposites (ZIF-8/KIT-6, ZIF-8/KIT-6/Resveratrol and ZIF-8/MCM-41/Curcumin) were formulated. Anti-blastocystosis activity of the tested nanocomposites was both dose and time dependent. ZIF-8/KIT-6/Resveratol showed the maximum percentage of growth inhibition (~ 100%) at a concentration of 500 µg/ml after 5 h of exposure. More than 90% of blastocystosis cysts’ growth was significantly inhibited at all concentrations of ZIF-8/MCM-41/Curcumin, with different times of exposure, while it occurred only at the highest concentration of ZIF-8/KIT-6 (800 µg/ml). Using cheap, simple, reproducible and scalable techniques, we nano-formulated innovative bioactive nanocomposites, by incorporating the bioactive ZIF-8 (Zn2+ with imidazole), structured mesosilica and natural antioxidant compounds, curcumin or resveratrol, to generate multifunctional modalities. These eco-friendly, naturally based, safe, economical, biocompatible, and bioavailable nanocomposites are potential nanotherapeutics. The anti-blastocystosis results of these three nanocomposites indicate their potentially promising innovative and safe use as alternative Blastocystosis therapies.


Materials and methodology
Nanocomposites. Innovative bioactive naturally based polyphenols were nano-formulated, by accommodating ZIF-8, structured nano-mesosilica (MCM-41 or KIT-6) and the natural antioxidants, curcumin or resveratrol. ZIF-8 with textural properties of 1040 m 2 /g (Z1200, sigma Aldrich), was purchased and used as a composite with mesoporous silica. The hexagonal shaped nano-silica (MCM-41) and three-dimensional KIT-6 was synthesized using a hydrothermal technique. Cationic template cetyltrimethyl ammonium bromide (CTAB) and non-ionic template (P123) were used respectively to generate hexagonal and cubic pores for MCM-41 and KIT-6.

Synthesis of MCM-41 and KIT-6.
For synthesis of MCM-41; 10.6 g of sodium metasilicate (silica source) was taken and dissolved in 50 ml of distilled water and stirred vigorously for 1 h. After dissolution, the solution was added dropwise to 4.5 g of CTAB solution dissolved in 40 ml of distilled water. The mixture was further stirred for 2 h and then pH was adjusted to 10.5 using diluted sulphuric acid solution (4 N). The sol solution in milky form was hydrothermally treated at 140 °C for 12-24 h.
For synthesis of KIT-6; 4 g of P123 was dissolved in acidic HCl solution (2 M) and stirred for 1 h; then 4 g of n-butanol (co-solvent) was added along with 8.6 g of tetraethylorthosilicate (silica source) and stirred for 24 h. The mixture in polypropylene bottle was transferred to oven and hydrothermally aged at 100 °C for 24 h. The precipitate was filtered, washed several times with excess water and dried at 100 °C for 12 h. The two samples (MCM-41 and KIT-6) were finally calcined at 550 °C for 6 h.
Drug release study. The  www.nature.com/scientificreports/ 7.4). The release of antioxidants was studied at 37 °C. At regular time intervals, 5 ml of solution was withdrawn, and release of antioxidants were measured using UV-visible spectroscopy. The withdrawn solution (5 ml) was replaced with equal volume of fresh PBS solution.
The release content was identified at the specific wavelength of curcumin and resveratrol (307 nm). Prior to analysis, calibration curve for curcumin and resveratrol was established. At first, an initial stock solution was prepared with concentration of 1000 µg/ml for curcumin and resveratrol. Various concentration of aliquots 5, 10, 15, 20, 25 and 30 µg/ml were prepared with makeup volume of 10 ml using release medium PBS solution (pH = 5.6 or 7.4) and calibration curve established against blank at maximum absorption wavelength λmax of 428 nm and 307 nm, respectively. Linear regression for curcumin and resveratrol were found to be y = 0.0041x + 0.0237 and y = 0.0579x + 0.0318, where y corresponds to absorbance and x to the concentration of antioxidant release (µg/ ml). The correlation coefficient was of 0.9982 and 0.995 for maximum absorption for curcumin and resveratrol, respectively. The release study was repeated in triplicates.
Biological activities of newly formulated nanocomposites. blastocystosis parasite. Suspension of cultured blastocystosis from symptomizing patients was used for evaluating biological activities of the formulated nanocomposites. blastocystosis were obtained from the El-Badry Lab and were ethically approved by the Deanship of Scientific Research and Postgraduate Studies of Imam Abdulrahman Bin Faisal University under the Institutional Review Board (IRB) number (IRB# 2021-01-009).
blastocystosis were sub-cultured using fresh Jones' medium enriched with 10% horse serum according to Jones 27 to get rid of stool debris 28 . Cultures with vacuolar forms of blastocystosis greater than 10 6 /ml were used for evaluating biological activities of the formulated nanocomposites.
Molecular characterization of isolated blastocystosis. One ml of subculture was suspended in 7 ml PBS, vortexed, centrifuged for 1 min at 12,000×g, then the pellet was kept for DNA extraction. DNA was extracted from pelleted blastocystosis cysts, using commercial DNA extraction kit (DNA MiniPrep™ kit, Zymo Research Corp., USA) following the kit's protocol. As per Stensvold's 29 recommendations, blastocystosis DNA was amplified and genotyped using two PCR reactions, targeting blastocystosis specific SSU rDNA 30 and subtype-specific Sequence-Tagged-Site (STS) 31 in order to detect the seven standardized subtypes (STs 1-7). PCR conditions and reactions were performed as mentioned by Yoshikawa et al. 30 and Scicluna et al. 31 .
In vitro challenging of blastocystosis with nanocomposites. Suspension of cultured blastocystosis cysts, in logarithmic growth phase of 10 6 /ml, were inoculated in sets of culture tubes with Jones' culture media. Haemocytometer counting chamber was used to count the number of blastocystosis cysts. The experiments contained 3 groups as follows: Group 1 (G1): parasite (negative, not treated) control group, containing only cultured cysts of blastocystosis. Group 2 (G2): drug (positive) control, containing cultured parasites exposed to 500 μg/ml metronidazole as a reference for anti-blastocystosis therapy. Group 3 (G3): nanocomposites tested group, containing cultured parasites exposed to the three nanocomposites. G3 was divided into subgroups to test three nanocomposites (G3A, G3B and G3C) at different concentrations (G3i-iv). Parasites were exposed to increasing concentrations of the three nanocomposites of 100 μg/ml (G3i) 200 μg/ml (G3ii), 500 μg/ml (G3iii) and 800 μg/ml (G3iv) (W/V). All tubes were incubated at 37 °C in humidified CO 2 for 48 h.
All tested nanocomposites for each concentration were prepared by diluting stock suspension in appropriate amount of PBS. The volume of each of the experiment tubes was brought to 1 ml with blastocystosis 10 6 /ml concentration. All experiments were performed in triplicate [32][33][34] .
Assessment of anti-blastocystosis activities of the nanocomposites. The number of blastocystosis cysts from all cultured tubes included in the experiment were counted under the microscope in haemocytometer counting chamber, and cultured blastocystosis cysts were tested for their viability using Trypan blue solution (0.4%). All cultured tubes were examined for percentage of reduction in growth of blastocystosis cysts each hour for 5 h then after 24 h. The minimal lethal concentration (MLC) was determined to be the concentration at which no blastocystosis cysts were observed 32-34 . Statistical analysis. The obtained data was statistically analyzed using SPSS software and presented as mean and standard deviation (SD). All data shown represents the mean for each of the three independent experiments. Means were compared and variances were analyzed. P values of less than 0.05 indicate statistical significance. ZIF-8 exhibited typical crystalline peaks corresponding to sodalite structure (Fig. 1a). Resveratrol and Curcumin indicated a characteristics crystalline peak (Fig. 1b,c). Four samples of composites were analyzed to study the extent of crystallite transformation of ZIF-8 and bioactive components in contact with mesoporous silica. The characteristic peaks of ZIF-8 and curcumin were not observed in XRD spectra of MCM-41/ZIF-8/ Curcumin and KIT-6/ZIF-8/Curcumin (Fig. 1d,e). These findings indicate the smaller crystallite size of ZIF-8 and the nanosize transformation of curcumin at the hexagonal and cubic pores of MCM-41 and KIT-6, respectively. In case of MCM-41/ZIF-8/Resveratrol, some peaks of resveratrol were seen, while in cubic shaped KIT-6 none were observed (Fig. 1f,g). These results show that some crystalline forms of resveratrol were still present inside the hexagonal pores of MCM-41. The transformation of ZIF-8 and antioxidants from crystalline to smaller crystallite size increased their bioavailability. The textural properties of MCM-41, KIT-6, MCM-41/ZIF-8 and KIT-6/ZIF-8 nanocomposites were measured using nitrogen adsorption technique ( Fig. 2Aa-d). The surface area, pore volume and average pore size values are presented in Table 1. The adsorption-desorption isotherm  Figure 3A shows the FTIR spectra of Resveratrol, ZIF-8, MCM-41, MCM-41/ZIF-8/Resveratrol and KIT-6/ ZIF-8/Resveratrol nanocomposites. The spectra of resveratrol and ZIF-8 showed characteristic bands corresponding to carbon-carbon double bonds of aromatic compound at 1605, 1583 and 1514 cm −1 (Fig. 3Aa,b). Phenolic compound containing a carbonyl group showed a band at 1155 cm −1 . The hydroxyl group from the phenolic compound showed a stretching at 1390 cm −1 . The C-H group showed a band at 960 cm −1 , indicating the trans resveratrol configuration. The C-H vibration band of arene conjugated to olefinic group can be seen at 805 cm −1 and 836 cm −1 . In the various bands that are observed between 650-500 cm −1 , a =C-H of olefinic group can be seen at 670 cm −1 . Compared to functional bands of hexagonal pores of MCM-41 (Fig. 3Ac), various functional bands of resveratrol in reduced signals were observed, indicating the presence of an effective surface functionalization at the external pores (Fig. 3Ad); however, no such bands of resveratrol were observed in the cubic shaped pores of KIT-6 ( Fig. 3Ae). This trend shows an effective pore filling of nanosized resveratrol in cubic pores of KIT-6. Figure 3B shows the FTIR spectra of curcumin, MCM-41/ZIF-8/Curcumin and KIT-6/ZIF-8/Curcumin nanocomposites (Fig. 3Ba-c). The functional groups of curcumin including > C=O and C=C were observed between 1627-1450 cm −1 . In particular, a characteristic enolic OH of curcumin can be observed at 962 cm −1 . The -C-O-C-chain vibrations (symmetric and asymmetric) can be observed between 1000-1450 cm −1 (Fig. 3Ba). In the case of both nanocomposites, the curcumin functional group showed a reduction in > C=O and C=C bands. A reduction in hydroxyl band of enol group also indicates an overall effective interaction of curcumin through enol and other functional groups (Fig. 3Bb,c)  KIT-6/ZIF-8 shows a strong absorption at 212 nm, which can be ascribed to the presence of Zn 2+ species in ZIF-8 (Fig. 4a). Resveratrol and curcumin revealed broad absorption between 200-600 nm (Fig. 4b,c). After loading of curcumin and resveratrol, the absorption maximum increases significantly over MCM-41/ZIF-8 and KIT-6/  www.nature.com/scientificreports/ ZIF-8/nanocomposites. Such expansion behavior clearly indicates the composite formation over two supports ( Fig. 4d-g).
Drug delivery study. The drug release ability of antioxidants on MCM-41/ZIF-8/Resveratrol and KIT-6/ ZIF-8/Resveratrol nanocomposites was studied at intestinal parasite pH condition for human intestine, ranging in pH from 5.3 to 7.4 (Fig. 5). Resveratrol (pure form) was studied for comparative purpose. In the release study, as-such resveratrol in the absence of nanocarrier showed a burst release as expected and reached a maximum of 43% within 12 h. In the case of nanocomposite/resveratrol release profiles, MCM-41/ZIF-8 exhibited a slow release of about 15% for 96 h, while KIT-6/ZIF-8 showed even a lower release profile of about 7% for 96 h. At neutral pH condition, both nanocomposites showed a reduced release of resveratrol. Figure 5f shows the curcumin on KIT-6 support alone. A quick release of curcumin was observed reaching 37% within 3 h and reached 52% within 96 h. In case of nanocomposites, a wider difference was observed in curcumin release. KIT-6/ZIF-8 showed a higher percentage of cumulative release of curcumin (39%) than MCM-41/ZIF-8 (21%) at pH 5.6. blastocystosis-DNA was amplified using the primers targeting blastocystosis specific SSU rDNA sequence of blastocystosis STs. blastocystosis DNA was amplified and genotyped as subtype-3 using the primers targeting blastocystosis subtype-specific STS. www.nature.com/scientificreports/ The anti-blastocystosis cysts activities of the three nanocomposites, ZIF-8/MCM-41/Curcumin, ZIF-8/KIT-6/ Resveratrol and ZIF-8/KIT-6 were determined by calculating the viability percent of blastocystosis cysts exposed to different increasing concentrations of all the nanocomposites each hour for 5 h then after 24 h (100, 200, 500 and 800 µg/ml). The parasite control group showed no effect on viability of blastocystosis cysts, while there was a reduction in number of cysts exposed to the nanocomposite; the maximum effect was observed with ZIF-8/ MCM-41/Resveratrol (Figs. 6, 7, 8 and Table 2).
ZIF-8/KIT-6/Resveratrol showed the maximum percentage of blastocystosis cysts destruction (~ 100%) at concentration of 500 µg/ml after 5 h of exposure, as found using standard drug (metronidazole) with statistical significance.  Table 2). There was a statistical significance in low doses of the nanocomposites ( Table 2).
Trypan blue is a vital stain which does not stain a live blastocystosis cysts (Fig. 9), while it stains dead blastocystosis cysts blue (Fig. 9). The dead blastocystosis cysts were further confirmed by microscopic detection of cell wall disruption and destruction of internal structures.

Discussion
Nanocomponents are at the forefront of the promising and expanding field of nanotechnology. Due to their highly crystalline nature, the antioxidants resveratrol and curcumin, are insoluble in water, and consequently of reduced bioavailability and poorly absorbed when taken orally 1,35 . The advantage of using a nanocarrier (nano-silica) with nano-size pores is its ability to transform the bioactive components from an insoluble state (crystalline state) into a nanoform soluble state (amorphous state), thus increasing their bioavailability 1,8 .
In the current study, resveratrol and curcumin was nano-formulated and loaded to ZIF-8/KIT-6 and ZIF-8/ MCM-41, a metal organic framework (MOF) built with bioactive and biocompatible components ZIF-8 and silica. This crystallite size transformation of ZIF-8 and the antioxidants resveratrol and curcumin amorphous form, enhanced their biocompatibility and bioavailability. Curcumin and resveratrol built with the two bioactive components (ZIF-8/MCM-41 and ZIF-8/KIT-6), work synergistically as antiparasitic nanotherapeutics.
MOFs are shown to exhibit pH sensitive drug releases. The degradation of MOF at acidic, neutral, and basic conditions tends to influence drug release characteristics. In the current study, the nanocarrier showed high drug loading efficiency and pH sensitive release capability due to inherent anchoring nature of metal-oxygen clusters. The high release at acidic pH of 5.5 could be attributed to the protonation of drug leading to a higher release than at the neutral pH of 7.4 36,37 . The ZIF-8 nanocarrier with pH sensitivity has been reported to be beneficial against parasite protozoan Trypanosoma infection. At acidic pH of 4.5, a quick release of benznidazole was observed, while the trend changes to a sustained and longer release at pH 7.4 38 . In our case, the hydroxyl functionalization of curcumin and resveratrol with nanocomposites may be critical in protonation and might assist such release in acidic pH condition. Both MCM-41/ZIF-8 and KIT-6/ZIF-8 nanocomposites showed a reduction in surface area and hierarchical pore formations compared to MCM-41 and KIT-6. In parallel, an intermediate pH sensitive release with steady modulation of antioxidants occurs in KIT-6/ZIF-8 more than with KIT-6 and resveratrol.
To date, there is no full cure for Blastocystosis, an emerging parasitic disease 26,39 . Small doses of metronidazole (as of 10 μg/ml) have been used as in-vitro anti-blastocystosis therapy 40,41 ; however, several studies stated the need for higher doses up to 1 mg/ml [40][41][42][43] . In the current study, we used a higher dose of 500 μg/ml of metronidazole.
Resveratrol has shown promising anti-protozoal activity, but its anti-blastocystosis activity has yet to be evaluated. In the current study, we assessed for the first time the in vitro effects of resveratrol and ZIF-8/KIT-6 nanocomponents on the viability of blastocystosis cysts. The current study showed that there was dose-dependent, and time depended anti-blastocystosis cysts activity for the two assessed nanocomposites at low concentrations. The www.nature.com/scientificreports/ MLC of resveratrol nanocomponents was 100 μg/ml, which is a much smaller dose than higher doses previously tested 44 . Juan et al. 44 reported that the prolonged eating (for 28 days) of large doses of resveratrol (1.4 g), 1000 times the amount ingested daily by a human of 70 kg, caused no harm to rats when compared to rat control group. The current study tested the anti-blastocystosis activity of nanocomponents (curcumin, resveratrol, KIT-6 and ZIF-8/MCM-41), which were used in higher doses in previous studies and proven to be non-cytotoxic for human cells 9,[44][45][46] .
We nano-formulated innovative nanocomposites, using cheap, simple, reproducible, and scalable techniques. They are biocompatible, bioavailable, stabile in acidic environments, eco-friendly, safe and economical naturally based compounds that can be used as alternative anti-microbial therapeutics. Table 2. Reduction in the percentage of viability of blastocystosis cysts in parasite control group (negative control), after exposure to metronidazole (positive control), and after exposure to the three nanocomposites. Data are presented as the mean ± standard deviation (SD), *P < 0.05 is statistically significant, compared to the parasite control. www.nature.com/scientificreports/ Many studies have attributed the anti-protozoal activities of resveratrol and curcumin to reduced oxygen consumption by protozoa 9,47-49 , which may explain its mechanism of action as anti-blastocystosis. There are many advantages to the nano-scaling of resveratrol and curcumin. It enhances their therapeutic activities by inducing cellular stress responses at lower doses. This low dose adaptive response is due to its nano-size with large surface area to volume ratio 13 . Giving the drug in lower doses reduces toxicity 13 . Remarkably, with prolonged exposure, all nanocomposites killed blastocystosis cysts, this may be because nanocomposites accumulate in host cells and their action is sustained for a longer time.
In the current study, Trypan blue vital stain enabled easy differentiation between viable and non-viable cysts, similarly several previous studies reported the reliability of Trypan blue stain in counting the viability of blastocystosis 32,34,40,50 . The blastocystosis clinical isolates from symptomatic patients used for our experiment were blastocystosis subtype 3. This tested strain was susceptible to the anti-blastocystosis effects of the nanocomposites used, particularly resveratrol.
These nanocomposites are biocompatible, bioavailable, economical and are stabile in acidic environments, making them promising alternative anti-microbial therapeutics.
The obtained findings provide first time evidence that ZIF-8/KIT-6/Resveratrol in addition to curcumin has anti-blastocystosis activity and destroys blastocystosis cysts in vitro with small MLC. The encouraging anti-blastocystosis results for these three nanotherapeutics suggest that they are safe and innovative alternative blastocystosis therapies. Further research is required to test their in vivo effect and to assess the anti-microbial activities of the formulated nanocomposites against other microbes.